Targeted Lipidomic and Transcriptomic Analysis Identifies Dysregulated Renal Ceramide Metabolism in a Mouse Model of Diabetic Kidney Disease

Abstract

Both type 1 and type 2 diabetes are associated with altered lipid metabolism, which might in part contribute to debilitating complications such as diabetic kidney disease (DKD). Ceramides are bioactive sphingolipids that have been implicated in a variety of diseases as they can regulate cellular responses to stress and invoke a myriad of downstream signaling responses. To investigate a potential role of altered ceramide metabolism in DKD, we utilized a highly sensitive and specific mass spectrometry (MS) method to quantitatively measure species in plasma and kidney cortex from the C57BLKS db/db mouse model of DKD and littermate controls. Long-chain ceramides (C14:0, C16:0, C18:0, C20:0) and a glucosylceramide (Glu-Cer C18:0) were increased in diabetic mouse plasma, while long-chain (C14:0, C16:0, C18:0) and very-long-chain (C24:0, C24:1) ceramides and a glucosylceramide (Glu-Cer C16:0) were decreased in diabetic mouse kidney tissue. Kidney and plasma ceramide levels correlated to functional and histopathological features of DKD. Transcriptomic analysis of mouse kidney tissue revealed expression changes indicative of decreased ceramide synthesis (Degs2, Smpd2) and increased conversion to sphingosine (Acer2) and downstream sphingosine-1-phosphate signaling. Correlation analysis identified a negative relationship between plasma and kidney tissue levels of ceramide C16:0 and ceramide C24:1. Overall, the findings suggest a previously unrecognized role for ceramide metabolism in DKD.

Keywords: Ceramide; Diabetes; Diabetic kidney disease; Diabetic nephropathy; Glucosylceramide; Kidney; Lipidomics; Mass spectrometry.

Figure 1. Extracted Ion Chromatograms of the measured ceramide species by LC/MS

Individual ceramide species were quantified using LC/ESI-MS/MS in the MRM mode on an Agilent 6410 triple quadruple mass spectrometer. Extracted ion chromatograms and m/z transitions for the 12 endogenous ceramide species and the Cer C17:0 internal standard are shown. In the nomenclature (Cer C14:0), the number before the colon refers to length of the carbon chain and the number after the colon to the number of double bonds in the chain.

Figure 2. Ceramide levels in diabetic mouse plasma and kidney tissue

Individual ceramide species were measured by LC/ESI-MS/MS in plasma (A) and kidney tissue (B) from 24 wk old control (db/+) and diabetic (db/db) mice by MS. Data was log2 transformed, normalized by plasma volume (μl) or tissue weight (mg), and is expressed as the relative log fold change in diabetic mice compared to control. Box-and-whiskers plots display distributions of each metabolite. The length of the box defines the interquartile range (IQR) while the whiskers denote the maximum and minimum value. The line in each box represents the median. * p < 0.05. Pearson’s correlation was used to define a relationship between plasma and kidney tissue levels per mouse. Linear regression models for ceramide species with a significant correlation (p < 0.05) are shown (C–D). In the nomenclature (Cer C16:0), the number before the colon refers to length of the carbon chain and the number after the colon to the number of double bonds in the chain.

Figure 3. Ingenuity Pathway Analysis of ceramide synthesis and downstream signaling

Transcriptomic analysis of kidney cortex from 24 wk old db/db diabetic mice and db/+ control mice for genes involved in ceramide metabolism identified altered expression of genes in ceramide synthesis and downstream sphingosine-1-phosphate signaling. Ingenuity® Pathway Analysis was used to visualize changes in both the canonical ceramide biosynthesis pathway and the sphingosine-1-phosphate signaling pathway. Green = significantly downregulated, pink = significantly upregulated, gray = unchanged.